Victor3v 1420 multilabel counter plate reader

Intracellular ROS and GSH levels were measured in HaCaT cells using CM-H2DCFDA dye (Invitrogen/Molecular probes) and ThiolTracker^TM Violet (Invitrogen/Molecular Probes) respectively upon treatment with 0.005 Gy, 0.05 Gy and 0.5 Gy ICCM. For both the assays, the study was performed in black 96-well microplates (Nunc, Roskilde, Denmark) and the cells were seeded at the density of 1x10⁵ cells/ml in 100 μl of respective media. After 24 hours of plating, the cells were loaded with 2.5 μM CM-H2DCFDA dye and 20 μM ThiolTracker^TM Violet for 30 mins at 37 ^οC for both the assays separately. After incubation with dyes, plates were washed with PBS three times before treatment with 0 Gy, 0.005 Gy, 0.05 Gy and 0.5 Gy ICCM and left at 37°C, 5% CO₂ incubator. The fluorescence intensities were measured after 5 min, 30 min, 1 hour, 1.5, 2, 2.5, 5, 10, 15, 20 and 24 hours respectively. For ROS assay, a Tecan microplate reader (TECAN GENios, Grodig, Austria) was used with excitation and emission wavelengths set at 485 nm and 530 nm. For Glutathione assay, a VICTOR3V™1420 Multilabel Counter plate reader (Perkin Elmer, Buckinghamshire, UK) was used with excitation and emission wavelengths set at 405 nm and 535 nm.

Sulforhodamine B (SRB) assay was chosen to verify the cytotoxic potential of ATXII [isolated and purified as previously described (Jarolim et al. 2017 (link))]. SRB experiments were performed according to the protocol of Skehan and collaborators (Skehan et al. 1990 (link)). Accordingly, at the end of the incubation (ATXII 0.1–10 µM; solvent controls 0.1% or 0.2% DMSO), HT-29 and HCEC were rinsed with PBS, fixed with 50% trichloroacetic acid (TCA; 30 min, 4 °C), dried and stained with SRB solution (0.4% w/w diluted in 1% acetic acid, 1 h, RT). Cells were then rinsed with diluted acetic acid (1%) and bi-distilled water. Protein-bound SRB reagent was diluted with Tris (10 mM) and absorbance was measured at 570 nm with a Victor³V 1420 Multilabel Counter Plate Reader (PerkinElmer, Waltham, USA). Data are presented as the means ± standard error of the mean (SE) of at least three independent cell preparations measured in technical triplicates. Statistical analysis was performed with Origin Pro 9.1G (OriginLab, Northampton, USA) applying one-way ANOVA with Fisher test for pairwise comparison (threshold value p < 0.05).